Method of obtaining a binder to prepro-vasopressin or fragments thereof

ABSTRACT

Method of obtaining and/or verifying a binder to prepro-Vasopressin (SEQ ID NO. 1) or fragments thereof of at least 6 amino acids in length, including Copeptin (SEQ ID NO. 2), comprising at least one of the steps of: a) generating the binder using a developer comprising an amino acid sequence of at least 6 amino acids in length contained in an amino acid sequence corresponding to the C-terminal part but lacking amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1); b) determining whether the binder is capable of binding to an amino acid sequence of at least 4 amino acids in length contained in an amino acid sequence corresponding to the C-terminal part but lacking amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1); c) selecting and optionally isolating the binder from a plurality of binders which is capable of binding to an amino acid sequence contained in an amino acid sequence corresponding to the C-terminal part but lacking amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1); d) carrying out binding assays with the binder in order to determine the ex vivo stability of prepro-Vasopressin or fragments thereof of at least 6 amino acids in length, including Copeptin, in a biological sample; e) carrying out binding assays with the binder and another binder for comparison purposes in order to determine the concentration of prepro-Vasopressin or fragments thereof of at least 6 amino acids in length, including Copeptin, in a biological sample; wherein the C-terminal part consists of amino acids 138 to 164 of prepro-Vasopressin (SEQ ID NO. 1), in order to obtain a binder or a mixture of binders capable of binding to an epitope contained in an amino acid sequence corresponding to amino acids 138 to 163 but lacking amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1).

The present invention relates to a method of obtaining and/or verifyinga binder to prepro-Vasopressin or fragments thereof, including Copeptin,a binder and a kit comprising the binder for qualitatively orquantitatively detecting prepro-Vasopressin or fragments thereof,including Copeptin, in a biological sample, and a peptide for generatinga binder to prepro-Vasopressin or fragments thereof, including Copeptin.

BACKGROUND OF THE INVENTION

Prepro-Vasopressin can be processed to Pro-Vasopressin by cleavage ofthe signal peptide, which can be further processed into Vasopressin,Neurophysin-2 and Copeptin, the latter representing the C-terminalmoiety of the precursor peptide. Vasopressin, also known as antidiuretichormone (ADH), is a key regulator of water and electrolyte balance. Themeasurement of Vasopressin in biological samples is barely possible inclinical routine due to considerable technical challenges related to itsrapid clearance from the circulation, interaction with platelets in theserum, and small size, see “Literature”, paragraph [0039], [1-3].Copeptin, which is stoichiometrically formed together with Vasopressin,has been successfully established as a surrogate marker for Vasopressin.A major reason for this success is considered its extremely high ex vivostability, making it suitable for routine use [4-6].

Clinical research has revealed numerous indications, where measurementof Copeptin gives highly useful diagnostic information, includingcardiological, pulmonological, infectious, kidney diseases, pathologicaldisturbances of the water and electrolyte balance, and others [5].Published methods for the detection of Copeptin are immunoassays [4, 6].

Prepro-Vasopressin and fragments thereof can be expressed alsoectopically in certain types of cancer, and anti-Copeptin antibodies canbe used to detect expression in tissue samples (EP 1539818 A2).

While immunogens for the generation of anti-Copeptin antibodies havebeen described, little, if any, is known about the actual epitopes ofthese antibodies. Described anti-human Copeptin antibodies are listed intable 1. In the prior art, no attention has been paid to the question onwhether and how the epitope specificity of anti-Copeptin antibodiesmight affect the accuracy and robustness of the detection ofprepro-Vasopressin or fragments thereof including Copeptin in biologicalsamples, using such antibodies. It is rather generally believed thatCopeptin is very stable per se. An antibody for detection of matureCopeptin, the epitope of which is rather unspecifically defined ashaving been mapped “near the C-terminus of Copeptin”, is offered bySanta Cruz Biotechnology, Inc., Heidelberg, Germany, and recommended foruse in ELISA and other applications.

TABLE 1 Antibody Origin Immunogen Epitope Reference Anti- SheepCATQLDGPAGALLLRLV Not described [6] PATV17representing positions 132-147 of pre-pro-Vasopressin plusan N-terminal cystein residue Anti- Sheep CLAGAPEPFEPAQPDAYNot described [6] PLAY17 representing positions 149-164of pre-pro-Vasopressin plus an N-terminal cystein residue 294/1A7 MouseATQLDGPAGALLLRLVQLAGAPEP- GPAGAL WO 2010049179 FEPAQPDAY representing A1representing positions 132-164 positions 137-144of pre-pro-Vasopressin plus of pre-pro-Vaso-an N-terminal cystein residue pressin H-065-32 RabbitASDRSNATQLDGPAGALLLRLVQLA- Not described Phoenix Pharma- GAPEPFEPAQPDAYceuticals, representing positions 126-164 Burlingame, USAof pre-pro-Vasopressin MAB6077 Mouse ASDRSNATQLDGPAG Not describedR+D Systems, (clone representing positions 126-140 Minneapolis, USA579021) of pre-pro-Vasopressin MAG-1 Mouse QLAGAPEPFEPAQPDAYNot described EP 1539818 A2 representing positions 148-164of pre-pro-Vasopressin sc-7811 Goat Not described “epitope mappingSanta Cruz Bio- near the C-termin- technology, Inc., us of Copeptin ofHeidelberg, Ger- human origin” many

DESCRIPTION OF THE INVENTION

The present invention relates to a method as claimed in claim 1, apeptide as claimed in claim 10 and the use thereof as claimed in claim11, a binder as claimed in claim 12, the use of said binder as claimedin claim 14, and a kit as claimed in claim 18. Preferred embodiments aredescribed in the respective dependent claims.

In detail, in a first aspect, the invention concerns a method ofobtaining and/or verifying a binder to prepro-Vasopressin (SEQ ID NO. 1)or fragments thereof of at least 6 amino acids in length, includingCopeptin (SEQ ID NO. 2), comprising at least one of the steps of:

-   a) generating the binder using a developer comprising an amino acid    sequence of at least 6 amino acids in length contained in an amino    acid sequence corresponding to the C-terminal part but lacking amino    acid 164 of prepro-Vasopressin (SEQ ID NO. 1);-   b) determining whether the binder is capable of binding to an amino    acid sequence of at least 4 amino acids in length contained in an    amino acid sequence corresponding to the C-terminal part but lacking    amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1);-   c) selecting and optionally isolating the binder from a plurality of    binders which is capable of binding to an amino acid sequence    contained in an amino acid sequence corresponding to the C-terminal    part but lacking amino acid 164 of prepro-Vasopressin (SEQ ID NO.    1);-   d) carrying out binding assays with the binder in order to determine    the ex vivo stability of prepro-Vasopressin or fragments thereof of    at least 6 amino acids in length, including Copeptin, in a    biological sample;-   e) carrying out binding assays with the binder and another binder    for comparison purposes in order to determine the concentration of    prepro-Vasopressin or fragments thereof of at least 6 amino acids in    length, including Copeptin, in a biological sample;    wherein the C-terminal part consists of amino acids 138 to 164 of    prepro-Vasopressin (SEQ ID NO. 1),    in order to obtain a binder or a mixture of binders capable of    binding to an epitope contained in an amino acid sequence    corresponding to amino acids 138 to 163 but lacking amino acid 164    of prepro-Vasopressin (SEQ ID NO. 1).

The main aspect of the present invention is the surprising finding thatfragments of prepro-Vasopressin and especially Copeptin in biologicalsamples are not extremely stable per se, as the prior art suggests, butthat analyte stability and the accurate and reliable detection depend onthe epitope against which antibodies used in an assay to detect thefragments, such as Copeptin, are directed. In particular, it was foundthat the exclusion of amino acid position 164 in the C-terminal portionof prepro-Vasopressin as part of the epitope for a binder toprepro-Vasopressin or fragments thereof, such as Copeptin, is highlycritical concerning both, accurate detection and analyte stability.Accordingly, the method comprises steps making sure to find thosebinders which do not require the presence of amino acid 164 in theepitope of an amino acid sequence corresponding to the C-terminal partof prepro-Vasopressin (SEQ ID NO. 1), i.e., the amino acid sequencestarting with and downstream of amino acid 138. As was found andconfirmed in the present invention, binders not requiring amino acid 164in the epitope for binding lead to more accurate and reliable analyticalresults and are thus more suited to be used in assays for detectingprepro-Vasopressin or fragments thereof than the binders needing saidamino acid 164 for binding.

It could further be shown that not only amino acid 164 may be lackingfrom the C-terminal portion of prepro-Vasopressin as part of the epitopefor a binder to prepro-Vasopressin or fragments thereof but thatimproved results may also be obtained when more than the one amino acidis missing. In more detail, amino acids 163 and 164, preferably 162-164and most preferably amino acids 161-164 may be lacking from the aminoacid sequence corresponding to the C-terminal part of prepro-Vasopressin(SEQ ID NO. 1). That is, the binder obtainable by the method of theinvention does not require the presence of these amino acids in theepitope for binding to prepro-Vasopressin or fragments thereof,including Copeptin (SEQ ID NO. 2).

The terms “prepro-Vasopressin” and “Copeptin” as used herein comprisealso amino acid sequences showing e.g. only 75% homology, preferred atleast 80% homology, more preferred at least 90% homology toprepro-Vasopressin and Copeptin, respectively. The same applies to otherfragments of prepro-Vasopressin than Copeptin. “Fragments” ofprepro-Vasopressin relate to fragments of at least 6 amino acids inlength, preferably at least 8, especially preferably at least 10 andmost preferably at least 12 amino acid residues in length.

The method of the invention describes several ways of obtaining and/orverifying suitable binders not needing amino acid 164 for binding, asdescribed above, which may be employed solely or in combination witheach other. A first possibility is given in step a) and concerns thepurposeful and selective generation of the binder by using a suitabledeveloper selectively leading to the desired binder. Concretely, thedeveloper comprises an amino acid sequence contained in an amino acidsequence corresponding to the C-terminal part but lacking amino acid 164of prepro-Vasopressin (SEQ ID NO. 1). That is, the developer comprisesan amino acid sequence of at least 6 amino acids in length whichcorresponds to at least a part of the amino acid sequence from aminoacids 138 to 164 of prepro-Vasopressin but does not have amino acid 164.Using a developer wherein amino acid 164 is missing in the amino acidsequence leads to a binder or a mixture of binders not requiring saidamino acid 164 for binding. (In the following, the term “binder” shallmean both a single type of binder or a mixture of different types ofbinders, unless otherwise stated.) Principally, step a) thus selectivelyand directly leads to the desired binder and does not require additionalsteps in order to remove binders needing said amino acid 164 forbinding. However, this does not exclude that, after step a), additionalsteps, such as characterization, purification, selection and isolationsteps, may follow.

In the context of this invention, the amino acid sequence contained inan amino acid sequence corresponding to the C-terminal part but lackingamino acid 164 of prepro-Vasopressin (SEQ ID NO. 1) used for generatingthe binder can principally be a synthetically prepared or naturallyderived amino acid sequence. It may be linear or folded. The said aminoacid sequence may principally be of any length of 6 or more amino acidssuitable for generating the desired binder or for obtaining efficientbinding to the binder. It may principally correspond to any amino acidsequence between amino acids 138 and 163 of prepro-Vasopressin.Preferably, the amino acid sequence is contained in the sequence ofamino acids 140 to 163, more preferably 142 to 163, especiallypreferably 144 to 163 and most preferably 146 to 163 ofprepro-Vasopressin (SEQ ID NO. 1). Suitably, the amino acid sequence hasat least 8, preferably at least 10 and most preferably at least 12 aminoacids. Particularly, said amino acid sequence contains at least 6,preferably at least 8, more preferably at least 10 and most preferablyat least 12 consecutive amino acids contained in the amino acid sequencecorresponding to amino acids 146 to 163 of prepro-Vasopressin (SEQ IDNO. 1).

As used herein, the term “binder” refers to any substance capable ofbinding to an epitope contained in an amino acid sequence correspondingto the C-terminal part of prepro-Vasopressin (SEQ ID NO. 1). This binderis generally shaped adequately, both spatially and in terms of surfacefeatures, such as surface charge, hydrophobicity, hydrophilicity,presence or absence of Lewis donors and/or acceptors, to specificallybind the target molecules or molecules of interest, i.e.,prepro-Vasopressin or fragments thereof, including Copeptin. Hereby, thebinding may for instance be mediated by ionic, van-der-Waals, pi-pi,sigma-pi, hydrophobic or hydrogen bond interactions or a combination oftwo or more of the aforementioned interactions between the binder andthe target molecules or molecules of interest. Such binders may beselected from but are not limited to the group consisting of antibodiesand aptamers.

Preferably, the binder is an antibody, preferably a monoclonal antibody,polyclonal antiserum, enriched or purified polyclonal antibody,recombinant antibody, or a functional derivative thereof. The term“antibody” as used herein, unless indicated otherwise, is used broadlyto refer to both, antibody molecules and a variety of antibody-derivedmolecules. Such antibody-derived molecules comprise at least onevariable region (either a heavy chain or a light chain variable region),as well as individual antibody light chains, individual antibody heavychains, chimeric fusions between antibody chains and other molecules,and the like. Functional immunoglobulin fragments according to thepresent invention may be Fv, scFv, disulfide-linked Fv, Fab, andF(ab′)2. Also encompassed by the term “antibody” are polyclonalantibodies, monoclonal antibodies, preferably IgG1 antibodies; chimericmonoclonal antibodies; humanized antibodies, genetically engineeredmonoclonal antibodies. Functional derivatives are chemically and/orbiochemically modified variants of the antibodies/antisera having ananalogous functionality/binding capacity. Analogously, the developer maybe any substance suited for generating a binder of the invention.Preferably, the developer is an immunogen, most preferably a natural orsynthetic peptide, comprising an amino acid sequence of at least 6 aminoacids in length contained in the amino acid sequence corresponding tothe C-terminal part of prepro-Vasopressin (SEQ ID NO. 1).

The term “developer” as used herein refers to a binding-site relatedsubstance for the generation of a binder, e.g. an amino acid sequence.The developer may consist of the amino acid sequence as described aboveor may contain this amino acid sequence as the functional part of thecompound. For example, the developer may additionally comprise a linkingpart, such as another amino acid sequence. Preferably, the developer isselected from the group comprising the peptides consisting of aminoacids 146-163 (SEQ ID NO. 7), 146-162 (SEQ ID NO. 8), 146-161 (SEQ IDNO. 9), 146-160 (SEQ ID NO. 10), 146-159 (SEQ ID NO. 11), 146-158 (SEQID NO. 12) and 146-157 (SEQ ID NO. 13) of prepro-Vasopressin (SEQ ID NO.1). These peptides and their use in the method of the invention are alsopart of the present invention. The methods of raising binders,particularly antibodies, using peptides are generally known in the artas thus need not be described here.

In step b) of the instant invention it is determined whether the binderis capable of binding to an amino acid sequence contained in an aminoacid sequence corresponding to the C-terminal part but lacking aminoacid 164 of prepro-Vasopressin (SEQ ID NO. 1). Here, and also in stepc), the amino acid sequences and the C-terminal part are defined as instep a) above, with the exception that a length of at least 4 aminoacids in the amino acid sequence is regarded as sufficient fordetermining binding. Preferably, the amino acid sequence has at least 6,more preferably at least 8, even more preferably at least 10 and mostpreferably at least 12 consecutive amino acids contained in the aminoacid sequence corresponding to amino acids 146 to 163 ofprepro-Vasopressin (SEQ ID NO. 1), and preferably is an amino acidsequence selected from the group consisting of amino acids 146-163 (SEQID NO. 7), 146-162 (SEQ ID NO. 8), 146-161 (SEQ ID NO. 9), 146-160 (SEQID NO. 10), 146-159 (SEQ ID NO. 11), 146-158 (SEQ ID NO. 12) and 146-157(SEQ ID NO. 13) of prepro-Vasopressin (SEQ ID NO. 1).

Step b) may, for example, be used in addition to step a) in order toverify the suitability of the binder obtained in the generating step.However, more preferably, step b) may be used in order to find outwhether a binder, which has not been prepared using a selectivedeveloper as in step a) and where it is not clear whether it will needsaid amino acid 164 in the epitope for binding, is actually capable ofbinding to an amino acid sequence of at least 4 amino acids in lengthcontained in an amino acid sequence corresponding to the C-terminal partbut lacking amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1). Thisalso applies to mixtures of binders where it may be necessary to findout whether it contains binders which require amino acid 164 in theepitope for effective binding. If this is confirmed these undesiredbinders may then be removed in a later step.

Step b) can be carried out using any suitable determining method knownto the expert. A preferred way of determining whether the binder iscapable of binding to an amino acid sequence of at least 4 amino acidsin length corresponding to the amino acid sequence of the C-terminalpart of prepro-Vasopressin is epitope mapping. Epitope mapping is aprocess known to a person skilled in the art. Epitope mapping is theprocess of identifying the binding sites, or “epitopes”, of binders ontheir target antigens. There are two types of general structures thatbinders use to bind antigens: linear and conformational. Linear epitopesare formed by a continuous sequence of amino acids in a protein, whileconformational epitopes are composed of amino acids that arediscontinuous in the protein sequence but are brought together uponthree-dimensional protein folding. There are several methods availablefor mapping binding epitopes on target antigens. The gold standardapproach is X-ray co-crystallography, which allows direct visualizationof the interaction between the antigen and the binder. However, thisapproach is technically challenging, requires large amounts of purifiedprotein, and can be time-consuming and expensive. An alternativeapproach for epitope mapping is peptide scanning. This technique uses alibrary of short peptide sequences from overlapping segments of a targetprotein and tests for their ability to bind the antibody of interest.This method is faster and relatively inexpensive, but is primarily usedfor mapping linear, not conformational, epitopes. In order to testwhether the binder is capable of binding to the respective amino acidsequence, the amino acid sequence can be bound directly or indirectly toa solid phase. For indirect binding the amino acid sequence may containthis amino acid sequence as the functional part of the compound. Forexample, the amino acid sequence may additionally comprise a linkingpart, such as biotin. Biotinylated amino acid sequences can beimmobilized via streptavidin, avidin, neutravidin or captavidin that isbound directly on the solid phase. As biotin binds with an extremelyhigh affinity and specificity to streptavidin, avidin, neutravidin orcaptavidin, the amino acid sequence that has to be tested for binding ofthe respective binder is consequently indirectly bound to the solidphase via a biotin/streptavidin-complex. Another approach for epitopemapping is site-directed mutagenesis. Using this approach, systematicmutations of amino acids are introduced into a protein sequence followedby measurement of specific binding of the binder in order to identifyamino acids that comprise an epitope. This technique has the advantageof mapping both linear and conformational epitopes, but islabor-intensive and slow, typically limiting analysis to a small numberof amino acid residues. All these methods can be used in step b) of theinvention. Preferably, however, in this process, binding of the binderagainst variants of the binding region is assessed. Such variants may betruncated, mutated, extended or otherwise modified representations ofthe binding region, which are typically produced by chemical synthesisor as recombinant peptides/proteins by molecular biological methods. Ithas to be noted that the interpretation of experimental epitope mappingdata may be strongly influenced by the experimental conditions applied,such as for instance the amount of binding targets offered,concentrations of binder applied, applied detection method, appliedincubation conditions etc. This is however known to the expert and canthus be taken into account.

If it has turned out—for example, in the determining step b) describedabove—that a mixture of binders was obtained, or if it is clear that thepreparation method leads to a mixture of binders, and the mixturecontains undesired binders which require amino acid 164 in the epitopefor effective binding, these undesired binders should be removed or atleast depleted. For this purpose, the method of the invention comprisesstep c) in which the desired binder not requiring amino acid 164 ofprepro-Vasopressin in the epitope for binding and thus leading toimproved analytical results is selected so that it may be separated fromthe undesired binder. This selecting step may be any process suited toselectively get hold of the desired binder in order to enrich itcompared with the undesired binder. A preferred way to do so is affinityseparation. This method uses the different binding properties of themolecules to be separated. The most common process is affinitychromatography wherein a ligand specific for the binding site of thetarget molecule is coupled to an inert chromatography matrix. Underbinding conditions this specific ligand on the chromatography matrixwill bind molecules according to its specificity only. All other samplecomponents will pass unbound through the chromatography medium. After awash step the bound molecules may then be released and eluted bychanging the conditions towards dissociation or by adding an excess of asubstance that displaces the target molecule from the affinity ligand(competitive elution). The binder can thus be isolated in purified form.The invention is, however, not restricted to the above selection,purification and isolating steps, but any other suitable methods canalso be employed.

Finding out whether a binder requires amino acid 164 ofprepro-Vasopressin in the epitope for binding may also be accomplishedby carrying out binding assays, according to steps d) and e) of theinvention. Both steps indirectly characterize the epitope specificity ofthe binder and are thus steps verifying the suitability of the binder.

As mentioned herein, an “assay” can be of any type applied in the fieldof diagnostics. Such an assay may be based on the binding of an analyteto be detected to one or more capture probes with a certain affinity.Concerning the interaction between capture molecules and targetmolecules or molecules of interest, the affinity constant is preferablygreater than 10⁸ M⁻¹. “Capture molecules” are molecules which may beused to bind target molecules or molecules of interest. The capturemolecules must thus be shaped adequately, both spatially and in terms ofsurface features, such as surface charge, hydrophobicity,hydrophilicity, presence or absence of Lewis donors and/or acceptors, tospecifically bind the target molecules or molecules of interest. Asmentioned before, the binding may for instance be mediated by ionic,van-der-Waals, pi-pi, sigma-pi, hydrophobic or hydrogen bondinteractions or a combination of two or more of the aforementionedinteractions between the capture molecules and the target molecules ormolecules of interest.

The assays can have various formats, such as for instanceradioimmunoassay (RIA), chemiluminescence- andfluorescence-immunoassays, Enzyme-linked immunoassays (ELISA),Luminex-based bead arrays, protein microarray assays, and rapid testformats such as for instance immunochromatographic strip tests.

The assays can be homogenous or heterogeneous assays, competitive andnon-competitive assays. In a particularly preferred embodiment, theassay is in the form of a sandwich assay, which is a non-competitiveimmunoassay, wherein the molecule to be detected and/or quantified isbound to a first antibody and to a second antibody. The first antibodymay be bound to a solid phase, e.g. a bead, a surface of a well or othercontainer, a chip or a strip, and the second antibody is an antibodywhich is labeled, e.g. with a dye, with a radioisotope, or a reactive orcatalytically active moiety. The amount of labeled antibody bound to theanalyte is then measured by an appropriate method. The generalcomposition and procedures involved with “sandwich assays” arewell-established and known to the skilled person (The ImmunoassayHandbook, Ed. David Wild, Elsevier LTD, Oxford; 3rd ed. (May 2005),ISBN-13: 978-0080445267; Hultschig C et al., Curr Opin Chem Biol. 2006February; 10(1):4-10. PMID: 16376134, incorporated herein by reference).In a particularly preferred embodiment the assay comprises two capturemolecules, preferably antibodies which are both present as dispersionsin a liquid reaction mixture, wherein a first labeling component isattached to the first capture molecule, wherein said first labelingcomponent is part of a labeling system based on fluorescence- orchemiluminescence-quenching or amplification, and a second labelingcomponent of said marking system is attached to the second capturemolecule, so that upon binding of both capture molecules to the analytea measurable signal is generated that allows for the detection of theformed sandwich complexes in the solution comprising the sample.

Even more preferred, said labeling system comprises rare earth cryptatesor rare earth chelates in combination with fluorescence dye orchemiluminescence dye, in particular a dye of the cyanine type.

In step d), a binding assay is carried out with the binder in order todetermine the ex vivo stability of prepro-Vasopressin or fragmentsthereof, including Copeptin, in a biological sample. As has been shownin the present invention, the analyte stability is higher when a binderis used not requiring amino acid 164 of prepro-Vasopressin or itsfragment for binding compared to those binders needing said amino acid164 for binding. Consequently, the analyte stability can be used to makeconclusions regarding the type of binder. Those analytes having thehighest stability are those not requiring amino acid 164 for binding andare thus the binders preferred in the present invention. Binders alreadyknown not to require amino acid 164 for binding can be used forcomparison purposes, i.e., the analyte stability related to thesecomparison binders can be used as a standard with which the analytestability evaluated for new binders is compared.

Step e), which may be used as an alternative or additional to step d),also uses another binder for comparison purposes, and binding assays arecarried out with this binder for comparison purposes and the new binderin order to determine the concentration of prepro-Vasopressin orfragments thereof, including Copeptin, in a biological sample. Thebinder for comparison purposes suitably is a binder already known to notrequire said amino acid 164 of prepro-Vasopressin for binding to thesaid epitope. A new binder also not needing said amino acid 164 ofprepro-Vasopressin in the epitope for binding is expected to giveresults with similar or higher concentrations of prepro-Vasopressin orfragments thereof, including Copeptin, in a biological sample as thebinder for comparison purposes, while binders requiring said amino acid164 are expected to result in lower concentrations.

As already mentioned above, the method of the invention may be comprisedof only one of steps a) to e). For example, it may consist solely ofstep a), step b), step c), step d) or step e). The method, however, alsoencompasses combinations of two or more of steps a) to e) in anysuitable order. For example, a preparation step suspected to havingresulted in a mixture containing both desired and undesired binders maybe followed by an analyzing step, such as step b), d) or e), which maythen optionally be followed by selecting the desired binders inaccordance with step c) and then, if so desired, by an isolating stepresulting in the enriched desired binder (which, as mentioned above, mayalso be a mixture of desired binders).

With the method of the invention, employing at least one of the methodsteps a) to e), it is possible to obtain those binders capable ofefficiently binding to an epitope contained in an amino acid sequencecorresponding to amino acids 138 to 163 but lacking amino acid 164 ofprepro-Vasopressin (SEQ ID NO. 1), which, as the invention hassurprisingly shown, are those binders which do not require the presenceof amino acid 164 in the epitope of an amino acid sequence correspondingto the C-terminal part of prepro-Vasopressin for effective binding. As aresult, more reliable analytical results can be obtained when using thebinder, which is obtainable by the method of the invention, or a kitcomprising said binder for qualitatively or quantitatively detectingprepro-Vasopressin or fragments thereof, including Copeptin, in abiological sample.

The biological sample may be any kind of bodily fluid and is preferablyselected from the group comprising blood, serum, plasma, urine,cerebrospinal fluid and saliva. Preferably, the sample is a bloodsample, most preferably a serum sample or a plasma sample. Whereappropriate, the sample may need to be homogenized, or extracted with asolvent prior to use in the present invention in order to obtain aliquid sample. A liquid sample hereby may be a solution or suspension.Liquid samples may be subjected to one or more pre-treatments prior touse in the present invention. Such pre-treatments include, but are notlimited to dilution, filtration, centrifugation, concentration,sedimentation, precipitation, dialysis. Pre-treatments may also includethe addition of chemical or biochemical substances to the solution, suchas acids, bases, buffers, salts, solvents, reactive dyes, detergents,emulsifiers, chelators.

“Plasma” in the context of the present invention is the virtuallycell-free supernatant of blood containing anticoagulant obtained aftercentrifugation. Exemplary anticoagulants include calcium ion bindingcompounds such as EDTA or citrate and thrombin inhibitors such asheparinates or hirudin. Cell-free plasma can be obtained bycentrifugation of the anticoagulated blood (e.g. citrated, EDTA orheparinized blood) for at least 15 minutes at 2000 to 3000 g. Therefore,it is preferred that plasma samples employed in the context of thepresent invention have been subjected to centrifugation at more than1500 g for 30 min, preferably at least at 2000 g for at least 30 min,more preferably at least at 3000 g for at least 20 min, most preferablyat least at 3000 g for at least 30 min.

“Serum” in the context of the present invention is the undiluted,extracellular portion of blood after adequate coagulation is completed.Coagulation is usually completed after 30 min. Serum can be obtained bycentrifugation of the coagulated sample for at least 10 minutes at aminimum speed of 1500 g. Therefore, it is preferred that serum samplesemployed in the context of the present invention have been subjected tocentrifugation at least at 1500 g for at least 10 min, preferably for atleast 15 min, more preferably for at least 20 min. Most preferably theserum sample has been subjected to a centrifugation at least at 3000 gfor at least 20 min.

The reliability of the results in determining prepro-Vasopressin orfragments thereof may be even more improved by using at least one otherbinder in addition to the binder of the invention. This other bindersuitably binds to another epitope of prepro-Vasopressin or its fragmentsthan the binder of the invention. The epitope used by the other binderfor binding preferably is an epitope fully or partially contained in anamino acid sequence corresponding to amino acids 126 to 164 ofprepro-Vasopressin (SEQ. ID NO. 1). Especially preferably, the epitopeis fully or partially contained in an amino acid sequence correspondingto amino acids 126 to 146 and most preferably in amino acids 126 to 137of prepro-Vasopressin. “Partially contained” in this context means thatonly a part of the epitope lies within the amino acid sequencecorresponding to amino acids 126 to 164 of prepro-Vasopressin while theother part lies upstream, towards the N-terminal, of the amino acidsequence. That is, part of the epitope overlaps with said amino acidsequence of prepro-Vasopressin while the rest of the epitope liesupstream thereof. The overlap preferably is at least 6 amino acids. Theadditional binder refers to any substance capable of binding to anepitope fully or partially contained in an amino acid sequencecorresponding to amino acids 126 to 164 of prepro-Vasopressin (SEQ IDNO. 1). Preferably, the additional binder is an antibody, preferably amonoclonal antibody, polyclonal antiserum, enriched or purifiedpolyclonal antibody, recombinant antibody, or a functional derivativethereof.

The invention will further be described with reference to the attacheddrawings and examples. The figures and examples relate to preferredembodiments of the invention but the invention is not restricted tothese embodiments but comprises all other embodiments encompassed by thescope of the claims.

DRAWINGS

FIG. 1 Epitope mapping of anti-PLAY17 sheep antiserum (FIG. 1 (A)),affinity purified sheep polyclonal anti-PLAY17 antibody (FIG. 1 (B)),mAb 429/F4 (FIG. 1 (C)) and mAb 423/F10 (FIG. 1 (D)). Data arerepresented as binding obtained against the respective indicated peptideminus non-specific binding (that is binding obtained, when the antibodyto be tested was omitted), relative to the binding obtained against theP146-164 peptide minus non-specific binding. Results are shown fordifferent dilutions/amounts of the anti-sera/antibodies tested.

FIG. 2 Dose response curves for the assays Pc anti-PLAY17/mc anti-PATV17(FIG. 2 (A)), mAb 429/F4/mc anti-PATV17 (FIG. 2 (B)), mAb 423/F10/mcanti-PATV17 (Fi. 2 (C)).

FIG. 3 Correlation of serum samples measured with Pc anti-PLAY17/mcanti-PATV17 and mAb 429/F4/mc anti-PATV17 (FIG. 3 (A)), Pcanti-PLAY17/mc anti-PATV17 and mAb 423/F10/mc anti-PATV17 (FIG. 3 (B)),mAb 423/F10/mc anti-PATV17 and mAb 429/F4/mc anti-PATV17 (FIG. 3 (C)).

FIG. 4 Correlation of EDTA-plasma samples measured with Pcanti-PLAY17/mc anti-PATV17 and mAb 429/F4/mc anti-PATV17 (FIG. 4 (A)),Pc anti-PLAY17/mc anti-PATV17 and mAb 423/F10/mc anti-PATV17 (FIG. 4(B)), mAb 423/F10/mc anti-PATV17 and mAb 429/F4/mc anti-PATV17 (FIG. 4(C)).

FIG. 5 Analyte stability. Shown are mean values (SEM) of five serumsamples after storage at 22° C. for the indicated time periods in therelation to the values measured without having the samples stored (t=0),when samples were measured with the assays indicated.

EXAMPLES Peptides

The following Copeptin-related peptides were chemically synthesized,purified, and quality controlled employing standard procedures:

SEQ Amino acid posi- ID tion of prepro- Peptide Sequence NO. VasopressinPAY16 CAGAPEPFE PAQ PDAY  4 150-164 (+ N-terminal Cystein) PAY14CAPEPFE PAQ PDAY  3 152-164 (+ N-terminal Cystein) P146-164LVQLAGAPEPFEPAQPDAY  6 146-164 P146-163 LVQLAGAPEPFEPAQPDA  7 146-163P146-162 LVQLAGAPEPFEPAQPD  8 146-162 P146-161 LVQLAGAPEPFEPAQP  9146-161 P146-159 LVQLAGAPEPFEPA 11 146-159 P146-158 LVQLAGAPEPFEP 12146-158 P146-157 LVQLAGAPEPFE 13 146-157

Antibodies

Monoclonal antibodies directed against the peptides PAY16 and PAY14 weregenerated by standard procedures (Harlow E, Lane D. Antibodies—ALaboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory,1988; Lane RD. A short-duration polyethylene glycol fusion technique forincreasing production of monoclonal antibody-secreting hybridomas. JImmunol Methods 1985; 81:223-8.):

Briefly, peptides were conjugated to BSA by using Sulfo-MBS(m-maleimidobenzoyl-N-hydroxysuccinimid ester). With these conjugatesBalb/c mice were immunized and boostered, and spleen cells were fusedwith SP2/0 myeloma cells to generate hybridoma cell lines. Cell lineswere screened for their ability to secrete antibodies that would bind tothe immunogenic peptides, which were coated on a solid polystyrenephase.

With this approach, cell lines secreting monoclonal antibodies 429/F4(against PAY16) and 423/F10 (against PAY14) were generated. For furtherexperiments, monoclonal antibodies were purified from culturesupernatant by Protein G affinity chromatography.

Sheep antiserum and corresponding affinity purified polyclonal sheepantibodies developed against peptide PLAY17 (“pc anti-PLAY”), used inchemiluminescence/coated tube assays to detect Copeptin (CT-proAVP) asdescribed [4, 7] were from BRAHMS GmbH, Hennigsdorf, Germany.

Epitope Mapping

The epitopes of the antibodies were mapped as follows:

a) Coating of Peptides

Coating was done by standard procedures (EP 1488209 A1, EP 1738178 A1):Polystyrene startubes (Greiner) were coated with peptides P146-164,P146-163, P146-162, P146-161, P146-159, P146-158 and P146-157 (per tube,1.5 μg of peptide in 300 μL of PBS, pH 7.8) overnight at 22° C. Tubeswere then blocked with 10 mmol/L Na-phosphate (pH 6.5) containing 3%Karion FP (Merck), 0.5% BSA protease free (Sigma) and lyophilized.

b) Labeling of Donkey-Anti-Sheep IgG and Goat-Anti-Mouse IgG Antibodies

Labeling was done by standard procedures (EP 1488209 A1, EP 1738178 A1):The concentration of the donkey-anti-sheep (Scantibodies LaboratoryInc., USA) and goat-anti-mouse antibody (BiosPacific, USA) was adjustedto 1 g/L, and the antibodies were labeled by incubation with thechemiluminescent label MACN-Acridinium-NHS-Ester (1 g/L; InVent GmbH,Hennigsdorf, Germany) in a 1:4 molar ratio for 20 min at roomtemperature. The reactions were stopped by addition of 1/10 volume of 1mol/L Tris for 10 min at room temperature. Labeled antibodies wereseparated from free label by size-exclusion chromatography on a NAP-5column (GE Healthcare, Freiburg, Germany) and a Thermo BioBasic 300 5 μmHPLC column (Thermo Scientific).

c) pc Anti-PLAY17 Antiserum/Affinity Purified Antibody

Tracer was produced by diluting the labeled donkey-anti-sheep IgGantibody in assay buffer PBS, 0.5% bovine serum albumin protease free(Sigma) containing 106 relative light units (RLU) of MACN-labeledantibody per 200 μl. Pc anti-PLAY17 sheep antiserum (B.R.A.H.M.S GmbH,Hennigsdorf, Germany) was diluted with PBS, 0.5% bovine serum albumin(protease free) at a ratio of 1:1000, 1:3000, 1:9000, 1:27000 and1:81000. Affinity purified pc anti-PLAY17 sheep antibodies were dilutedwith PBS, 0.5% bovine serum albumin protease free to followingconcentrations: 972, 324, 108, 36 and 12 ng/200 μl. In the firstincubation step 50 μl of the dilutions of pc anti-PLAY17 sheepantiserum/purified antibodies and 200 μl PBS, 0.5% bovine serum albumin(protease free) were pipetted in tubes, coated with peptides P146-164,P146-163, P146-162, P146-161, P146-159, P146-158 and P146-157. Forcalculation of non-specific binding (NSB) only 250 μl PBS with 0.5%bovine serum albumin (protease free) were pipetted in tubes, coated withpeptides P146-164, P146-163, P146-162, P146-161, P146-159, P146-158 andP146-157. The tubes were incubated over night at 22° C. under agitation.Then, the tubes were washed 5 times with 1 mL of B.R.A.H.M.S washingsolution (B.R.A.H.M.S GmbH). In the second incubation step 200 μl ofdonkey-anti-sheep IgG tracer were added and the tubes were incubated for2 hours at 22° C. under agitation. Then, the tubes were washed 5 timeswith 1 mL of B.R.A.H.M.S washing solution and bound chemiluminescencewas measured for 1 s per tube with an LB 952T luminometer (Berthold).

d) mAb 429/F4 and mAb 423/F10

Tracer was produced by diluting the labeled antibody goat-anti-mouse IgGinto assay buffer (PBS, 0.5% protease free bovine serum albumin)containing 106 relative light units (RLU) of MACN-labeled antibody per200 μl. Monoclonal antibodies 429/F4 and 423/F10 were diluted with PBS,0.5% bovine serum albumin (protease free) to the followingconcentrations: 972, 324, 108, 36 and 12 ng/200 μl.

In the first incubation step 50 μl of the dilutions of mAb 429/F4/mAb423/F10 and 200 μl PBS, 0.5% bovine serum albumin (protease free) werepipetted in tubes, which were coated with peptides P146-164, P146-163,P146-162, P146-161, P146-159, P146-158 and P146-157. For calculation ofNSB, only 250 μl PBS, 0.5% bovine serum albumin (protease free) werepipetted in tubes, coated with peptides P146-164, P146-163, P146-162,P146-161, P146-159, P146-158 and P146-157. The tubes were incubated overnight at 22° C. under agitation. Then, the tubes were washed 5 timeswith 1 mL of B.R.A.H.M.S washing solution (B.R.A.H.M.S GmbH,Hennigsdorf, Germany). In the second incubation step 200 μl ofgoat-anti-mouse tracer were added and the tubes were incubated for 2hours at 22° C. under agitation. Then, the tubes were washed 5 timeswith 1 mL of B.R.A.H.M.S washing solution and bound chemiluminescencewas measured for 1 s per tube with an LB 952T luminometer (Berthold).

In FIGS. 1 (A) to 1 (D) the observed binding of the antiserum andantibodies to peptides representing C-terminally full-length andtruncated variants of the C-terminal part of Copeptin is shown.Anti-PLAY17 sheep antiserum, affinity purified sheep polyclonalanti-PLAY17 antibody and mAb 429/F4 exhibited comparable binding topeptides corresponding to amino acid positions 146-164, 146-163,146-162, 146-161 of prepro-Vasopressin. With peptide variants, whichwere C-terminally more truncated, binding was reduced. The amount ofreduction was dependent on the concentrations of the antibodies applied.For the antisera/antibodies tested it is concluded that their epitopesdo not contain amino acid positions 161-164 of prepro-Vasopressin. Incontrast, binding of mAb 423/F10 was only efficient against a peptidecorresponding to amino acid positions 146-164 of prepro-Vasopressin, andwas strongly reduced against C-terminally truncated peptide variants.Thus, in the epitope of mAb 423/F10 amino acid position 164 ofprepro-Vasopressin is contained.

Immunoassays Labeling of Monoclonal Antibodies

Labeling was done by standard procedures (EP 1488209 A1, EP 1738178 A1):The concentration of the purified antibodies 429/F4 and 423/F10 wasadjusted to 1 g/L, and the antibodies were labeled by incubation withthe chemiluminescent label MACN-Acridinium-NHS-Ester (1 g/L; InVentGmbH, Hennigsdorf, Germany) in a 1:5 molar ratio for 20 min at roomtemperature. The reactions were stopped by addition of 1/10 volume of 1mol/L Tris for 10 min at room temperature. Labeled antibodies wereseparated from free label by size-exclusion chromatography on an NAP-5column (GE Healthcare, Freiburg, Germany) and a Thermo BioBasic 300 5 μmHPLC column (Thermo Scientific).

Three sandwich immunoassays were utilized or developed as follows:

A. Pc anti-PLAY17/mc anti-PATV17

CT-proAVP LIA (B.R.A.H.M.S GmbH, Hennigsdorf, Germany) as described in(71.

B. mAb 429/F4/mc anti-PATV17

Tracer was produced by diluting the labeled antibody 429/F4 into assaybuffer (300 mmol/L potassium phosphate, 100 mmol/L NaCl, 10 mmol/Lsodium EDTA, 5 g/L protease free bovine serum albumin, 1 g/L nonspecificsheep IgG, 1 g/L nonspecific bovine IgG, 1 g/L nonspecific mouse IgG,0.9 g/L sodium azide, pH 7.0) containing 106 relative light units (RLU)of MACN-labeled antibody per 200 μl. 50 μl CT pro-AVP standards(B.R.A.H.M.S GmbH, Hennigsdorf, Germany)/samples and 200 μl of tracerwere pipetted in CT pro-AVP coated tubes (B.R.A.H.M.S GmbH). The tubeswere incubated for 2 hours at 22° C. under agitation. Then, the tubeswere washed 5 times with 1 mL of washing solution (B.R.A.H.M.S GmbH),and bound chemiluminescence was measured for 1 s per tube with an LB952T luminometer (Berthold).

C. mAb 423/F10/mc anti-PATV17

Tracer was produced by diluting the labeled antibodies 423/F10 intoassay buffer (300 mmol/L potassium phosphate, 100 mmol/L NaCl, 10 mmol/Lsodium EDTA, 5 g/L protease free bovine serum albumin, 1 g/L nonspecificsheep IgG, 1 g/L nonspecific bovine IgG, 1 g/L nonspecific mouse IgG,0.9 g/L sodium azide, pH 7.0) containing 106 relative light units (RLU)of MACN-labeled antibody per 200 μl. 50 μl CT pro-AVP standards(B.R.A.H.M.S GmbH, Hennigsdorf, Germany)/samples and 200 NI of tracerwere pipetted in CT pro-AVP coated tubes (B.R.A.H.M.S GmbH). The tubeswere incubated for 2 hours at 22° C. under agitation. Then, the tubeswere washed 5 times with 1 mL of washing solution (B.R.A.H.M.S GmbH),and bound chemiluminescence was measured for 1 s per tube with an LB952T luminometer (Berthold).

Typical dose response curves for the three assays are shown in FIG. 2.

Method Comparison

With the three assays described above, various clinical samples weremeasured, including samples from healthy individuals, patients withcardiological diseases and patients from the ICU. Graphical methodcomparisons are shown in FIG. 3 (sera) and FIG. 4 (EDTA-plasma samples).Ideal Spearman correlation coefficients were observed, when assay pcanti-PLAY17/mc anti-PATV17 was compared with assay mAb 429/F4/mAbPATV17, whereas clear differences were observed, when assay mAb423/F10/mc anti-PATV17 was compared to pc anti-PLAY17/mc anti-PATV17 orassay mAb 429/F4/mc anti-PATV17. The differences were more pronouncedwith sera than with EDTA-plasma samples. The differences observed areclearly associated with the epitopes of the antibodies: The epitope ofmAb 423/F10 contains amino acid position 164 of prepro-Vasopressin,whereas the epitopes of pc anti-PLAY17 and mAb 429/F4 do not containamino acid positions 161-164 of prepro-Vasopressin. Apparently, besidesfull-length Copeptin there are also C-terminally truncated variantspresent in both, serum and plasma. As the samples used in the methodcomparison had been frozen immediately after harvesting and were thawedimmediately prior to its measurement, the partial C-terminal truncationapparently had not occurred as a consequence of ex vivo storage, but hadoccurred already in vivo.

Analyte Stability

A subset of serum samples used in the method comparisons was stored fordifferent periods at 22° C. and then measured with the three assays. Byusing the mAb 423/F10/mc anti-PATV17 assay, the recovery droppeddrastically, already after 1 day storage at 22° C. The epitope of mAb423/F10 contains amino acid position 164 of prepro-Vasopressin. Incontrast, the analyte appeared much more stable when either the pcanti-PLAY17/mc anti-PATV17 or mAb 429/F4/mc anti-PATV17 assay was used.The epitopes of pc anti-PLAY17 and mAb 429/F4 do not contain amino acidpositions 161-164 of prepro-Vasopressin.

Sequences

(prepro-Vasopressin) SEQ ID NO. 1        10         20         30         40MPDTMLPACF LGLLAFSSAC YFQNCPRGGK RAMSDLELRQ        50         60         70         80CLPCGPGGKG RCFGPSICCA DELGCFVGTA EALRCQEENY         90       100        110        120LPSPCQSGQK ACGSGGRCAA FGVCCNDESC VTEPECREGF        130       140        150        160HRRARASDRS NATQLDGPAG ALLLRLVQLA GAPEPFEPAQ PDAY (Copeptin) SEQ ID NO. 2ASDRSNATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY(representing amino acid positions 126-164 of prepro-Vasopressin)(Peptide PAY14) SEQ ID NO. 3 CAPEPFEPAQPDAY(representing amino acid positions 152-164 of prepro-Vasopressinplus an N-terminal cysteine) (Peptide PAY16) SEQ ID NO. 4CAGAPEPFEPAQPDAY (representing amino acid positions150-164 of prepro-Vasopressin plus an N-terminal cysteine)(Peptide PAY33) SEQ ID NO. 5 ATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY(representing amino acid positions 132-164 of prepro-Vasopressin)(Peptide P146-164) SEQ ID NO. 6 LVQLAGAPEPFEPAQPDAY(representing amino acid positions 146-164 of prepro-Vasopressin)(Peptide P146-163) SEQ ID NO. 7 LVQLAGAPEPFEPAQPDA(representing amino acid positions 146-163 of prepro-Vasopressin)(Peptide P146-162) SEQ ID NO. 8 LVQLAGAPEPFEPAQPD(representing amino acid positions 146-162 of prepro-Vasopressin)(Peptide P146-161) SEQ ID NO. 9 LVQLAGAPEPFEPAQP(representing amino acid positions 146-161 of prepro-Vasopressin)(Peptide P146-160) SEQ ID NO. 10 LVQLAGAPEPFEPAQ(representing amino acid positions 146-160 of prepro-Vasopressin)(Peptide P146-159) SEQ ID NO. 11 LVQLAGAPEPFEPA(representing amino acid positions 146-159 of prepro-Vasopressin)(Peptide P146-158) SEQ ID NO. 12 LVQLAGAPEPFEP(representing amino acid positions 146-158 of prepro-Vasopressin)(Peptide P146-157) SEQ ID NO. 13 LVQLAGAPEPFE(representing amino acid positions 146-157 of prepro-Vasopressin)

LITERATURE

-   1. Kluge M, Riedl S, Erhart-Hofmann B, Hartmann J, Waldhauser F.    Improved extraction procedure and RIA for determination of    arginine-8-vasopressin in plasma: role of premeas-urement sample    treatment and reference values in children. Clin Chem    1999;45:98-103.-   2. Preibisz J J, Sealey J E, Laragh J H, Cody R J, Weksler B B.    Plasma and platelet vasopressin in essential hypertension and    congestive heart failure. Hypertension 1983;5:1129-38.-   3. Robertson G L, Mahr E A, Athar S, Sinha T. Development and    clinical application of a new method for the radioimmunoassay of    arginine vasopressin in human plasma. J Clin Invest 1973;52:2340-52.-   4. Morgenthaler N G, Struck J, Alonso C, Bergmann A. Assay for the    measurement of co-peptin, a stable peptide derived from the    precursor of vasopressin. Clin Chem 2006;52:112-9.-   5. Morgenthaler N G, Struck J, Jochberger 5, Dunser M W. Copeptin:    clinical use of a new biomarker. Trends Endocrinol Metab    2008;19:43-9.-   6. Struck J, Morgenthaler N G, Bergmann A. Copeptin, a stable    peptide derived from the vasopressin precursor, is elevated in serum    of sepsis patients. Peptides 2005;26:2500-4.-   7. Fenske W, Stork S, Blechschmidt A, Maier S G, Morgenthaler N G,    Allolio B. Copeptin in the differential diagnosis of hyponatremia. J    Clin Endocrinol Metab 2009; 94:123-9.

1. Method of obtaining and/or verifying a binder to prepro-Vasopressin(SEQ ID NO. 1) or fragments thereof of at least 6 amino acids in length,including Copeptin (SEQ ID NO. 2), comprising at least one of the stepsof: a) generating the binder using a developer comprising an amino acidsequence of at least 6 amino acids in length contained in an amino acidsequence corresponding to the C-terminal part but lacking amino acid 164of prepro-Vasopressin (SEQ ID NO. 1); b) determining whether the binderis capable of binding to an amino acid sequence of at least 4 aminoacids in length contained in an amino acid sequence corresponding to theC-terminal part but lacking amino acid 164 of prepro-Vasopressin (SEQ IDNO. 1); c) selecting and optionally isolating the binder from aplurality of binders which is capable of binding to an amino acidsequence contained in an amino acid sequence corresponding to theC-terminal part but lacking amino acid 164 of prepro-Vasopressin (SEQ IDNO. 1); d) carrying out binding assays with the binder in order todetermine the ex vivo stability of prepro-Vasopressin or fragmentsthereof of at least 6 amino acids in length, including Copeptin, in abiological sample; e) carrying out binding assays with the binder andanother binder for comparison purposes in order to determine theconcentration of prepro-Vasopressin or fragments thereof of at least 6amino acids in length, including Copeptin, in a biological sample;wherein the C-terminal part consists of amino acids 138 to 164 ofprepro-Vasopressin (SEQ ID NO. 1), in order to obtain a binder or amixture of binders capable of binding to an epitope contained in anamino acid sequence corresponding to amino acids 138 to 163 but lackingamino acid 164 of prepro-Vasopressin (SEQ ID NO. 1).
 2. The method ofclaim 1, wherein amino acids 163-164, preferably amino acids 162-164 andmost preferably amino acids 161-164 are lacking from the amino acidsequence corresponding to the C-terminal part of prepro-Vasopressin (SEQID NO. 1).
 3. The method of claim 1, wherein the amino acid sequencecorresponding to the C-terminal part of prepro-Vasopressin (SEQ IDNO. 1) corresponds to amino acids 140 to 163, preferably 142 to 163,especially preferably 144 to 163 and most preferably 146 to 163 ofprepro-Vasopressin (SEQ ID NO. 1).
 4. The method of any one of claim 1,wherein the amino acid sequence comprised in the developer and/or theamino acid sequence contained in the amino acid sequence correspondingto the C-terminal part of prepro-Vasopressin (SEQ ID NO. 1) contains atleast 8, preferably at least 10 and most preferably at least 12consecutive amino acids contained in the amino acid sequencecorresponding to amino acids 146 to 163 of prepro-Vasopressin (SEQ IDNO. 1) and is preferably selected from the group comprising the peptidesconsisting of amino acids 146-163 (SEQ ID NO. 7), 146-162 (SEQ ID NO.8), 146-161 (SEQ ID NO. 9), 146-160 (SEQ ID NO. 10), 146-159 (SEQ ID NO.11), 146-158 (SEQ ID NO. 12) and 146-157 (SEQ ID NO. 13) ofprepro-Vasopressin (SEQ ID NO. 1).
 5. The method of claim 1, wherein thebinder is capable of binding to an amino acid sequence of at least 6,preferably at least 8, more preferably at least 10 and most preferablyat least 12 consecutive amino acids contained in the amino acid sequencecorresponding to amino acids 146 to 163 of prepro-Vasopressin (SEQ IDNO. 1), preferably an amino acid sequence selected from the groupconsisting of amino acids 146-163 (SEQ ID NO. 7), 146-162 (SEQ ID NO.8), 146-161 (SEQ ID NO. 9), 146-160 (SEQ ID NO. 10), 146-159 (SEQ ID NO.11), 146-158 (SEQ ID NO. 12) and 146-157 (SEQ ID NO. 13) ofprepro-Vasopressin (SEQ ID NO. 1).
 6. The method of claim 1, wherein thedetermining step b) comprises epitope mapping.
 7. The method of claim 1,wherein the specificity of binding of the binder to the said epitope isdetermined, particularly, in steps d) and/or e), by comparison with theepitope specificity of another binder for comparison purposes,preferably a binder known not to require the said amino acid 164 ofprepro-Vasopressin (SEQ ID NO. 1) for binding to the said epitope. 8.The method of claim 1, wherein the selecting step comprises affinityseparation.
 9. The method of claim 1, comprising step a) but not stepsb), c), d) and e), or step b) but not steps a), c), d) and e), or stepc) but not steps a), b), d) and e), or step d) but not steps a), b), c)and e), or step e) but not steps a), b), c) and d), or steps b) and c)but not steps a), d) and e).
 10. A peptide, consisting of or containing,in addition to a linking part, as a functional part at least 6,preferably at least 8, more preferably at least 10 and most preferablyat least 12 consecutive amino acids contained in the amino acid sequencecorresponding to amino acids 146 to 163 of prepro-Vasopressin (SEQ IDNO. 1), preferably peptide selected from the group comprising thepeptides consisting of amino acids 146-163 (SEQ ID NO. 7), 146-162 (SEQID NO. 8), 146-161 (SEQ ID NO. 9), 146-160 (SEQ ID NO. 10), 146-159 (SEQID NO. 11), 146-158 (SEQ ID NO. 12) and 146-157 (SEQ ID NO. 13) ofprepro-Vasopressin (SEQ ID NO. 1).
 11. The use of a peptide of claim 10as a developer in the method of claim 1, wherein the peptide isconsisting of or containing, in addition to a linking part, as afunctional part at least 6, preferably at least 8, more preferably atleast 10 and most preferably at least 12 consecutive amino acidscontained in the amino acid sequence corresponding to amino acids 146 to163 of prepro-Vasopressin (SEQ ID NO. 1), preferably peptide selectedfrom the group comprising the peptides consisting of amino acids 146-163(SEQ ID NO. 7), 146-162 (SEQ ID NO. 8), 146-161 (SEQ ID NO. 9), 146-160(SEQ ID NO. 10), 146-159 (SEQ ID NO. 11), 146-158 (SEQ ID NO. 12) and146-157 (SEQ ID NO. 13) of prepro-Vasopressin (SEQ ID NO. 1).
 12. Abinder, obtainable by the method of claim
 1. 13. A binder according toclaim 12, namely antibody, preferably monoclonal antibody, polyclonalantiserum, enriched or purified polyclonal antibody, recombinantantibody, or a functional derivative thereof.
 14. A use of the binder ofclaim 12 for qualitatively or quantitatively detectingprepro-Vasopressin or fragments thereof, including Copeptin, in abiological sample.
 15. The use of claim 14, wherein the biologicalsample is a bodily fluid selected from the group comprising blood,serum, plasma, urine, cerebrospinal fluid and saliva.
 16. The use ofclaim 14, wherein at least one additional binder is utilized which iscapable of binding to an epitope fully or partially contained in anamino acid sequence corresponding to amino acids 126 to 164 ofprepro-Vasopressin (SEQ ID NO. 1).
 17. The use of claim 16, wherein theat least one additional binder is an antibody, preferably monoclonalantibody, polyclonal antiserum, enriched or purified polyclonalantibody, recombinant antibody, or a functional derivative thereof. 18.A kit for qualitatively or quantitatively detecting prepro-Vasopressinor fragments thereof, including Copeptin, in a biological sample,comprising the binder of claim 12.